Synthesis of polyesters containing disiloxane subunits: Structural characterization, kinetics, and an examination of the thermal tolerance of Novozym-435

Article abstract of DOI:10.1016/j.molcatb.2012.09.010

This paper reports the Novozym-435 mediated polymerization of disiloxane-containing polyester monomers under solvent-free conditions. The thermal tolerance of the immobilized enzyme was examined by conducting polymerization cycles over a temperature range of 35-150 °C. Increasing the temperature up to 100 °C afforded an increase in the apparent second order rate constant. Residual activity was measured using the production of octyl palmitate. The enzyme was shown to retain on average greater than 90% of its residual activity regardless of the polymerization temperature. This prompted a study of the long term thermal tolerance of the biocatalyst in which it was determined that over ten reaction cycles there was a significant decrease in the initial polymerization rate, but no change in the degree of monomer conversion after 24 h. The disiloxane containing polyesters were characterized using nuclear magnetic resonance spectroscopy and Fourier-transform infrared spectroscopy. Differential scanning calorimetry was used to determine the thermal properties of the disiloxane-containing polyesters.

Full text of DOI:10.1016/j.molcatb.2012.09.010

Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 149–155
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Synthesis of polyesters containing disiloxane subunits: Structural
characterization, kinetics, and an examination of the thermal tolerance of
Novozym-435
Mark B. Framptona,1, Jacqueline P. Séguina,1, Drew Marquardt , Thad A. Harroun , Paul M. Zelisko
b
b
a,∗
a
Department of Chemistry and Centre for Biotechnology, Brock University, St. Catharines, Ontario, Canada
b
Department of Physics, Brock University, St. Catharines, Ontario, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 February 2012
Received in revised form 15 August 2012
Accepted 12 September 2012
Available online 19 September 2012
This paper reports the Novozym-435 mediated polymerization of disiloxane-containing polyester
monomers under solvent-free conditions. The thermal tolerance of the immobilized enzyme was
◦
examined by conducting polymerization cycles over a temperature range of 35–150 C. Increasing the
◦
temperature up to 100 C afforded an increase in the apparent second order rate constant. Residual activ-
ity was measured using the production of octyl palmitate. The enzyme was shown to retain on average
greater than 90% of its residual activity regardless of the polymerization temperature. This prompted a
study of the long term thermal tolerance of the biocatalyst in which it was determined that over ten
reaction cycles there was a significant decrease in the initial polymerization rate, but no change in the
degree of monomer conversion after 24 h. The disiloxane containing polyesters were characterized using
nuclear magnetic resonance spectroscopy and Fourier-transform infrared spectroscopy. Differential scan-
ning calorimetry was used to determine the thermal properties of the disiloxane-containing polyesters.
Keywords:
Polyester
Silicones
Lipase
Biocatalysis
©
2012 Elsevier B.V. All rights reserved.
◦
◦
1
. Introduction
actually range from 90 to 180 . The siloxane linkage is highly polar-
isable as a result of the high ionic nature of the Si O bond which has
been determined to be approximately 51% ionic [3]. The Si O bond
possesses very low rotational bond energy as well as being one of
the strongest chemical bonds known with a covalent bond energy
of 452 kJ/mol (108 kcal/mol) and ionic bond energy of 1013 kJ/mol
(242 kcal/mol) [2].
Silicones and other siloxane-derived materials are of great
industrial and economic importance [1]. Low molecular weight
cyclic silicones are commonly found in the health care and cos-
metics industries where they are typically incorporated into topical
applications, the food and beverage industry where they are used
as antifoaming agents, and in sealants and coatings where their
inherent hydrophobicity is the desired characteristic.
The popularity of siloxanes is a result of their physical and
chemical properties, which makes them well suited for a range
of applications. Siloxanes, and in particular silicones, are valued
for their thermal stability (silicones typically do not degrade until
Many methods have been designed to produce linear, branched,
or cross linked silicones. Most commonly used are catalysts based
on the platinum complexes developed by Karstedt and Speier,
and on alkoxytitanium complexes. Tin carboxylates are com-
monly used, particularly in the room temperature vulcanization
of silicone elastomers. However, the potential toxicity of alkyl
tin complexes [4] renders this class of catalyst undesirable espe-
cially in the synthesis of biomaterials. Other general cure methods
include high temperature vulcanizations, and thermal- or copper-
mediated [1,3]-dipolar additions [5,6]. More recent work on the
◦
temperatures in excess of 350 C are obtained), low glass transition
temperatures which result from the dynamic nature of the siloxane
bond, resistance to oxidation, and low permittivity values [1,2]. The
basis for these physical characteristics is derived from the Si
O Si
bond. The angle of the siloxane linkage is rather larger, typically
Piers–Rubensztajn reaction has lead to the development of B(C F5)3
6
◦
◦
1
45 , compared to 109.5 for typical tetrahedral organic systems.
as a catalyst for the preparation of star-shaped siloxanes and func-
tionalized silicones from hydrosilanes and alkoxysilanes [7–9].
Polyesters are commercially available in a variety of products
such as fibres, fillings, coatings, and textiles and are typically pro-
It has been postulated that this angle may be flexible enough to
◦
duced under extremes of heat, sometimes in excess of 250 C, and
∗ Corresponding author. Tel.: +1 905 680 5550x4389; fax: +1 905 682 9020.
under reduced pressure [10]. A variety of catalytic methods have
been developed for producing polyester materials, typically using
E-mail address: pzelisko@brocku.ca (P.M. Zelisko).
Tel.: +1 905 680 5550x4389; fax: +1 905 682 9020.
1
1
381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.09.010

1
50
M.B. Frampton et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 149–155
strong acids over stoichiometric quantities of both diols and diacids.
Alternatively, dibutyltin oxide or dibutyltin dilaurate have been
employed, but given the aforementioned toxicity concerns, these
catalysts may wish to be avoided. While strong acids are ideal for
polyester synthesis, they are not always compatible with siloxane
polymers due to the possibility for redistribution of the siloxane
backbone, or cleavage of the siloxane network [1]. Additionally,
the condensation of diols with acyl chlorides, and the ring opening
polymerization of lactones have been explored as viable routes to
polyester synthesis [11].
Enzymatic methods are becoming increasingly popular in
organic, bioorganic, and polymer chemistry. Lipase B from Can-
dida antarctica immobilized on a macroporous acrylic resin and
sold under the trade name Novozym-435, has been the work horse
for synthesizing polymeric materials [12–15] and has been used
to synthesize organosilicon amides and esters [16]. Furthermore,
an Amberzyme-immobilized cutinase from Humicola insolens has
garnered some recent attention for its polyester synthase ability
Anachemia Science (Montréal, Québec, Canada). Distilled water
was used when necessary. Chemicals were used as received with-
out further modification or purification unless otherwise stated.
All of the commercially available reagents were at a minimum
pure by NMR (≥95%).
2
2
.2. Methods
.2.1. Nuclear magnetic resonance spectroscopy (NMR)
All NMR spectra were recorded in CDCl on a Bruker Avance
3
1
13
29
AV-300 spectrometer ( H at 300 MHz, C at 77 MHz, and Si at
9.6 MHz) using the residual signal of CHCl as an internal reference
for H spectra, and the three C resonances of CDCl as the internal
reference for C NMR spectra; tetramethylsilane (TMS) was used
as an internal standard for Si NMR spectra. NMR spectra were
5
3
1
13
3
13
29
analysed using the Bruker Topspin v2.0 software interface.
2.2.2. Fourier-transform infrared spectroscopy (FTIR)
[
17,18]. This cutinase possessed stricter substrate specificity than
FTIR spectra were recorded on a Mattson research series infrared
N435 showing a preference for C10 and C13 diacids whereas those
diacids with chain lengths shorter than C10 were not processed
particularly well.
spectrometer operating in transmittance mode. Samples were
prepared as neat, thin films on KBr plates. Each spectrum was com-
−
1
prised of 32–64 scans at 2 cm
resolution. Analysis of the FTIR
The synthesis of polyesters incorporating siloxanes has been
demonstrated although, typically, the siloxane is only a minor com-
ponent of the final polymer system [13,19–22]. The enzymatic
synthesis of polyesters derived exclusively from siloxane-derived
monomers has been described [23]. The number of siloxane units
of the diol monomer did not affect the polymerization kinetics or
the activation energy of the polymerization process [23].
In this paper disiloxane polyesters were synthesized employing
N435 catalysis under solvent-free reaction conditions. The dis-
iloxane polyesters were subsequently characterized by nuclear
magnetic resonance (NMR) spectroscopy and Fourier-transform
infrared (FT-IR) spectroscopy. Differential scanning calorimetry
data was performed using the Winfirst software platform. Peak
assignments were made based on data previously reported in the
literature [24].
2.2.3. Differential scanning calorimetry (DSC)
DSC thermograms were acquired on a Shimadzu DSC-60 dif-
ferential scanning calorimeter. Polymer samples (approximately
10 mg) were transferred into aluminium pans and cooled to
◦
◦
◦
−150 C at a rate of 10 C/min. Samples were heated at 20 C/min
◦
◦
◦
to 200 C and subsequently cooled at 10 C/min to −150 C. A sec-
◦
◦
ond heating scan was done at 20 C/min to 200 C. DSC data was
analysed using the TA60 version 2.11 software platform.
(
DSC) was employed to determine the thermal transitions of the
monomers and resulting siloxane containing polyesters.
Synthesis
of
1,3-bis(3-hydroxypropyl)-1,1,3,3-
In order for biocatalysis to be viable on an industrial scale
cost must be minimized. One method to facilitate this would be
to design a catalyst with a high turnover number, or a catalyst
that would be amenable to multiple reaction cycles. The resid-
ual activity of N435 was examined, after each polymerization, by
using a standard enzymatic assay in which the production of octyl
palmitate from 1-octanol and palmitic acid was monitored. In con-
junction with the residual activity assays, a single batch of N435
tetramethyldisiloxane (4, 3HP-TMDS, Scheme 1). A round bottomed
flask was charged with 1,1,3,3-tetramethyldisiloxane (2) and
Karstedt’s catalyst and stirred for 10 min. Allyl acetate (1)
was added drop-wise through
a septum over 30 min and
the reaction mixture was allowed to reflux for 2 h to give
1,3-bis(3-acetoxypropyl)-1,1,3,3-tetramethyldisiloxane
(3)
1
in 83% isolated yield. H NMR (300 MHz, CDCl3, 7.26 ppm):
0.06, 0.11, 0.25 and 0.28 ppm (Me2SiO ), 0.5 ppm (m,
CH2CH2CH2Si, 4H), 1.63 ppm (m, CH2CH2CH2Si, 4H), 4.01 ppm (m,
◦
was used for multiple 24 h reaction cycles at 100 C to gain some
insight into the thermal tolerance and reusability of N435 as a poly-
merization catalyst.
AcOCH CH CH Si, 4H), CH C O (s, 3H); 13C NMR (77.0 MHz, CDCl3,
2
2
2
3
77.0 ppm): 0.22 ppm ((CH ) SiO ), 14.12 ppm (CH CH CH Si),
3
2
2
2
2
2
1.00 ppm (CH CH CH Si), 22.59 ppm (CH CO R), 66.94 ppm
2 2 2 2
3
(
AcOCH CH CH ), 171.16 ppm (C O). De-acylation of bis-
2 2 2
2
. Experimental
acetate 3 was routinely carried out using an 8 fold excess of
anhydrous MeOH and 20 mol% K CO3 for 2 h at room tem-
2
2.1. Materials
perature to yield diol 4 in nearly 87–95% yield as a clear to
straw coloured liquid [25]. 1H NMR (300 MHz, CDCl , 7.26 ppm):
3
Lipase B from C. antarctica immobilized on acrylic resin
0.05 ppm (s, 12H, Me Si), 0.528 ppm (m, 4H, CH CH CH Si),
2
2
2
2
(
1
sold under the trade name Novozym-435, N435; EC.3.1.1.3,
0,000 U/g), activated carbon, 1,1,3,3-tetramethyldisiloxane
1.646 ppm (m, 4H, CH CH CH Si), 2.327 ppm (t, 4H, CH CH CH Si,
2 2 2 2 2 2
J = 7.5 Hz); 13C NMR (77 MHz, CDCl , 77.01 ppm): 0.25 ppm (Me Si),
3 2
(
TMDS, 97%) and platinum(0)-1,3-divinyl-1,1,3,3-tetramethyl
18.03 ppm (CH CH CH Si), 19.12 ppm (CH CH CH Si), 37.48 ppm
2 2 2 2 2 2
0
29
disiloxane complex (Karstedt’s catalyst, Pt (dvs)) in xylenes were
obtained from Sigma–Aldrich (Oakville, Ontario, Canada). 1,3-
Bis(3-carboxypropyl)-1,1,3,3-tetramethyldisiloxane (CPr-TMDS)
was obtained from Gelest (Morristown, PA, USA). Allyl acetate
(
(
icals (Georgetown, Ontario, Canada). Chloroform-d (CDCl , 99.8%
deuterated) was a product of Cambridge Isotope Laboratories,
Inc. (Andover, MD, USA). Diethyl ether (99%) was acquired from
(CH CH CH Si);
EI-MS: (M ) 250 m/z.
Si NMR (59.6 MHz, CDCl , TMS): 7.28 ppm;
3
2
2
2
+
Synthesis
of
1,3-bis(3-carboxypropyl)-1,1,3,3-
tetramethyldisiloxane dimethyl ester (5, CPr-TMDS-DME). Diester
5 was synthesized using previously published protocols [23].
Briefly, 5.01 g (16.34 mmol) of 1,3-bis(3-carboxypropyl)-1,1,3,3-
tetramethyldisiloxane was refluxed in 15 mL of methanol in
the presence of 5 mol% p-toluene sulfonic acid for 4 h to yield
diester 5 as a clear and colourless liquid in 86% yield. 1H NMR
98%) was obtained from Alfa Aesar (Ward Hill, MA, USA). Isooctane
2,2,4-trimethylpentane, 99%) was obtained from Caledon Chem-
3

M.B. Frampton et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 149–155
151
Scheme 1. The synthesis of 1,3-bis(3-hydroxypropyl)-1,1,3,3-tetramethyldisiloxane.
(
(
300 MHz, CDCl , 7.26 ppm): 0.05 ppm (s, 12H, Me Si), 0.528 ppm
enzyme-free control reaction was also performed to determine the
background rate, if any, for the esterification of octyl palmitate.
3
2
m, 4H, CH CH CH Si), 1.646 ppm (m, 4H, CH CH CH Si),
2 2 2 2 2 2
2
.327 ppm (t, 4H, CH CH CH Si, J = 7.5 Hz), and 3.663 ppm(s, 6H,
2 2 2
1
3
MeOCO); C NMR (77 MHz, CDCl , 77.01 ppm): 0.25 ppm (Me Si),
2.2.6. Thermal tolerance and reuse of N435
3
2
1
8.03 ppm (CH CH CH Si), 19.12 ppm (CH CH CH Si), 37.48 ppm
A single batch of N435 was subjected to multiple 24 h reaction
cycles at 100 C to determine the number of times that the enzyme
2
2
2
2
2
2
◦
(
CH CH CH Si), 53.39 ppm (MeOCO), 174.09 ppm (MeOC O);
2
2
2
2
9
Si NMR (59.6 MHz, CDCl , TMS): 7.28 ppm (Si
O
Si); EI-MS:
could be reused before it was determined to be unsuitable for catal-
ysis. The apparent second order rate constant, which was derived
from the slope of the line from a plot of the average degree of
polymerization versus time, as well as the extent of monomer con-
version were compared. Between each 24 h reaction cycle, the N435
beads were recovered by the addition of 5 mL of diethyl ether at
room temperature for 10 min. After stirring, the beads were filtered
through a glass filter of medium porosity and washed with three
3
+
(
M −CH ) 319 m/z.
3
2.2.4. Preparation of disiloxane polyesters
Disiloxane polyesters were prepared under solvent-free con-
ditions using 5 wt.% N435 (with respect to the mass of the
combined monomers) following previously provided protocols
with minor modifications [23]. Typically, diol 4 was combined in
a 1:1 mole ratio with diester 5, using approximately 0.5–0.75 g
of the monomers. Individual reactions were incubated at temper-
1
0 mL aliquots of diethyl ether. The mass of the beads recovered
was determined and the volumes of each reagent for subsequent
reaction mixtures were adjusted accordingly.
◦
◦
atures between 35 C and 150 C with stirring for 24–72 h. The
reactions were terminated with the addition of 5 mL of diethyl
ether after cooling to RT. The enzyme beads were removed by fil-
tration through a glass fritted Buchner filter of medium porosity
and washed with two 10 mL volumes of diethyl ether. Solvents
were removed in vacuo to yield siloxane-polyesters as clear and
3
. Results and discussion
One of our research goals is the exploration of enzymatic
methods to mediate traditional organosilicon transformations. To
address these goals, we have employed enzymatic catalysis in the
form of the immobilized lipase B from C. antarctica (N435) for the
synthesis of disiloxane-containing materials. The reaction scheme
for the enzyme-mediated polymerization under investigation is
outlined briefly in Fig. 1.
colourless, viscous liquids. 1H NMR (300 MHz, CDCl , 7.26 ppm):
3
0.059 ppm (s, Me Si), 0.535 ppm (m, CH CH CH Si), 1.642 ppm
2 2 2 2
(
m, CH CH CH Si), 2.320 ppm (t, CH CH CH Si), and 3.667 ppm
2 2 2 2 2 2
13
(
7
s, MeOCO), 4.021 ppm (t, C(O)OCH2 ); C NMR (77 MHz, CDCl3,
7.01 ppm): 0.02 and 0.19 ppm (Me Si), 13.99 and 18.03 ppm
2
(
(
CH CH CH Si), 19.12 and 22.57 ppm (CH CH CH Si), 37.65 ppm
2 2 2 2 2 2
29
3.1. Structural characterization of disiloxane polyesters
CH CH CH Si), 66.6 ppm (CH OCO), 173.58 ppm (MeOC O); Si
2
2
2
2
NMR (59.6 MHz, CDCl , TMS): 7.30 and 7.72 ppm (Si
O Si).
3
The structural environments of the disiloxane-derived polymers
were probed primarily through NMR and FT-IR spectroscopies.
A typical 1H NMR spectrum for the disiloxane polyesters is pre-
sented in Fig. 2. There is a triplet positioned at 4.02 ppm which
represents the methylene protons alpha to the oxygen atom in
the newly formed ester linkage between the disiloxane-containing
monomers. There is a smaller singlet located at 3.67 ppm that is
assigned as unreacted methyl ester end groups [23]. The methy-
lene protons alpha to the carbonyl in diester 2 experience a small
shift up-field from 2.34 ppm in the monomer to 2.32 ppm in the
final polymer, while the O-methylene protons of diol 4, formally
positioned at 3.60 ppm were only barely visible. The remaining
multiplets, 0.54 ppm and 1.64 ppm, represent the methylene pro-
tons that are in the ␣- and ␤-positions with respect to silicon;
the geminal methyl groups on silicon are positioned at 0.06 ppm
and remained largely unchanged throughout the polymerization
process.
2.2.5. Residual enzyme activity of recovered lipase
The recovered N435 was assayed for residual activity by measur-
ing the production of octyl palmitate from 1-octanol and palmitic
acid. A stock solution of 200 mM 1-octanol and 200 mM palmitic
acid in isooctane was freshly prepared before each assay. For each
reaction 5 mg of lipase was combined with 1 mL of the assay mix-
◦
ture and incubated at 35 C for 1 h with magnetic stirring. A 20 ␮L
aliquot of the reaction mixture was analysed by 1H NMR. The ratio
of the resonances associated with the O-methylene protons in 1-
octanol and octyl palmitate was used to determine the degree of
esterification. The methylene resonance in the ester is located at
4
.02 ppm while the corresponding methylene resonance in the 1-
octanol is located at 3.66 ppm. A fresh enzyme preparation, that is
enzyme that had not been used to synthesize disiloxane polyester
systems, was prepared for use as a control. The amount of esteri-
fication achieved from the fresh enzyme was set to a default value
of 100% and all other assays were calibrated to this value. An
The 13C NMR spectrum confirms the synthesis of the disilox-
ane polyesters. The resonance for the carbonyl carbon resides

1
52
M.B. Frampton et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 149–155
Fig. 1. The synthesis of disiloxane polyesters using N435 under solvent-free conditions.
Fig. 2. A representative 1H NMR spectrum of enzymatically synthesized disiloxane polyesters.
◦
at 173.6 ppm while the O-methylene carbon can be found at
6.6 ppm; the carbon adjacent to the ester linkage was positioned
at 37.7 ppm. The remaining resonances were located at 13.99 ppm
and 17.96 ppm have been identified as CH CH Si (in monomer and
reheated at 10 C/min. The glass transition temperatures (Tg) were
acquired from the second heating scan. DSC thermograms for a rep-
resentative disiloxane polyester, along with diol 4 and diester 5, are
presented in Fig. 4. The Tg for diol 4 and diester 5 were determined
6
2
2
◦
◦
polymer), and 19.06 ppm and 22.57 ppm has been identified as
to be −99 C and −109 C, respectively. These values are somewhat
29
the CH CH Si environment in monomer. The Si spectrum pos-
higher than that for a typical polydimethylsiloxane which shows
2
2
◦
sessed two distinct resonances which were located at 7.30 ppm and
.72 ppm. These signals were in the expected range for disiloxane
a Tg near −125 C [2]. The disiloxane polyester 6 exhibited inter-
◦
7
mediate Tg values at −104 C. As a result of the dynamic nature of
linkages and do not represent a possible silanol or alkoxysilane.
The FTIR spectrum for a typical disiloxane polyester is pre-
sented in Fig. 3. The assignment of vibrational modes was made
based on previously reported data [24]. The carbonyl bond shows
the disiloxane linkage, well defined melting temperatures and cold
4000
3600
3200
2800
2400
2000
1600
1200
800
400
−
1
a strong absorbance located at 1737 cm
which was formerly
−1
located at 1742 cm in the methyl ester monomer. The charac-
teristic vibrational modes of the siloxane fragments are present as
−1
the Si
O
Si stretches located at 1050 cm . The FT-IR spectra sup-
Si
port the hypothesis that the enzyme did not participate in Si
O
cleavage as the expected vibrational stretching mode for the free
silanol (∼3350 cm−1) was not observed [26].
3.2. Thermal characteristics
The thermal characteristics of the disiloxane polyesters were
probed using differential scanning calorimetry (DSC). DSC thermo-
grams were acquired using a Shimadzu DC-60 differential scanning
calorimeter under air. Disiloxane-derived monomers, as well as
the enzymatically produced disiloxane polyesters, were cooled to
◦
◦
−
150 C from room temperature at a rate of −10 C/min. Each
◦
sample was then heated to 200 C and subsequently cooled and
Fig. 3. Representative FTIR spectra of a disiloxane-containing polyester.

M.B. Frampton et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 149–155
00
153
1
90
80
70
60
50
40
30
20
10
0
3
5
50
70
80
90
100
110
120
130
140
150
Temperature (ºC)
Fig. 4. Representative DSC thermograms of starting materials and the enzymat-
ically produced disiloxane polyesters. Top thermogram: CPr-TMDS-DME, middle
thermogram: 3HP-TMDS, and bottom thermogram: disiloxane polyester.
Fig. 6. Monomer conversion after 24 h of polymerization using N435 as catalyst.
Each bar is the average of multiple replicate trials; error bars represent the standard
deviation.
crystallization temperatures could not be determined for any of the
polyester samples.
thus forcing the equilibrium to favour the products in accordance
with Le Châtelier’s principle. Further increases in temperature
were hypothesized to be deleterious to the functioning of the
enzyme as a result of thermal denaturation. However, polymeri-
3.3. Disiloxane polymerization
◦
zations conducted at 100 C did not have the expected effect of
The synthesis of polyesters from the polymerization of diols,
protein denaturation. In fact the apparent rate constant associ-
ated with the polymerization continued to increase. To test the
thermal tolerance of N435, we continually increased the reaction
with diesters, diacids or acid chlorides is generally thought to be
a second order process [27]. The apparent rate constant for these
reactions can be determined using a plot of the average degree
of polymerization (DP) versus time, where DP = 1/(1 − p) in which
p is the extent of monomer conversion during the first hour of
the reactions. Enzyme-free control reactions were performed at all
temperatures and no background rate of transesterification was
observed. Our experiments showed that the reaction temperature
at which each polymerization was conducted had a significant
impact on the initial reaction rate (Fig. 5). At reaction tempera-
◦
temperature in ten degree intervals to a maximum of 150 C and
performed 24 h polymerization cycles. The expectation was that
at elevated temperatures the enzyme would become completely
and irreversibly denatured. To our surprise each batch of N435
polymerized the disiloxane monomers with increasing proficiency
◦
◦
reaching a maximum at 130 C; above 130 C the rate of poly-
merization slowly decreased. Furthermore, there appeared to be
a complete loss of catalytic activity when polymerizations were
◦
tures equal to or less than 70 C, the polymerization of monomers
◦
carried out at 150 C. These observations suggest that the optimal
◦
was slow while increasing the reaction temperature above 70 C
polymerization temperature of disiloxane containing monomers by
afforded higher polymerization rates. This increase can be par-
tially attributed to the ease of removal of the methanol by-product
◦
N435 is approximately 130 C, higher than that reported for the
polymerization of poly(caprolactone) and poly(pentadecalactone)
[
28].
The ratio of the resonances at ı 3.66 and 4.02 were compared
4
3
3
2
2
1
1
0
0
.0
.5
.0
.5
.0
.5
.0
.5
.0
to determine the degree of monomer conversion after 24 h of poly-
merization (Fig. 6). As the reaction temperature was increased from
◦ ◦
5 C to 90 C there was a steady increase in monomer conversion.
3
◦
For polymerizations conducted above 90 C, monomer conversion
seemed to plateau between 85% and 90%. We were concerned that
a potential cause for the apparent plateau was a reduced capacity
of the enzyme to process the disiloxane-containing substrates. This
hypothesis was tested by separating the enzyme from the reaction
mixture and assaying an aliquot of the enzyme for residual activity.
3.4. Residual activity of N435
The residual activity of the immobilized enzyme was quanti-
fied by monitoring the production of octyl palmitate from 200 mM
◦
1-octanol and 200 mM palmitic acid in isooctane at 35 C for 1 h
Fig. 7) [13]. A typical assay was performed using 5.0 mg of recov-
3
0
40 50 60 70 80 90 100 110 120 130 140 150 160
(
Temperature (°C)
ered lipase beads in 1 mL solution of the ester precursors; reactions
◦
were carried out at 35 C for 60 min with the aid of magnetic stir-
Fig. 5. The effect of temperature on the apparent second order rate constant for
the N435-catalysed polymerization of diol 4 with diester 5. Each data point is the
average of three trials; error bars represent the standard error of the means. The
line is included to guide the eye.
ring. Control assays which consisted of fresh N435 that had never
been used, and an enzyme-free control reaction were included for
comparison. Octyl-palmitate assays were monitored by 1H NMR

1
54
M.B. Frampton et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 149–155
Fig. 7. The esterification of 1-octanol and palmitic acid to octyl palmitate was used to determine the residual activity of N435 after each polymerization reaction.
again using the change in the intensity of the resonances for the O-
methylene protons in 1-octanol and octyl palmitate. In the absence
of any enzyme esterification between 1-octanol and palmitic acid
was not observed; no observable resonance visible at 4.02 ppm was
apparent. On the contrary, the virgin enzyme processed 93% (n = 3)
of the 1-octanol within the allotted time period. All subsequent
assays were normalized to this value to give the relative residual
activity. Every batch of recovered enzyme that was assayed, even
The change in the apparent second order rate constant and
monomer conversion for each of 10 successive uses of the same
batch of N435 is presented in Fig. 8. With the exception of the
first three trials in which the apparent rate constant remained con-
stant, each successive use of the immobilized enzyme led to some
loss in catalytic activity. The apparent rate constant decreased by
approximately 50% after six reaction cycles and by the tenth reac-
tion cycle more than 80% of the initial enzyme activity had been
lost. Despite the loss in catalytic activity, when the polymerizations
were allowed to continue for the full 24 h reaction cycle, monomer
conversion continued to reach high levels, typically in the range of
80–93%. These results can be compared to a previous study where
a single batch of N435 was used for the ring opening polymeriza-
tion (ROP) of ε-caprolactone to produce polycaprolactone (PCL) in
◦
those used at 100 C, retained in excess of 95% of their relative activ-
ity compared to the virgin enzyme. Binns et al. [29] reported the
residual activity of CALB to range between 6% and 88% after the
polymerization of adipic acid with a range of simple diols. In effect,
the residual activity of the enzyme increased with the decreasing
polarity and increasing hydrophobicity of the reaction medium. The
highest residual enzyme activity was seen when 1,5-pentane diol
and 1,6-hexane diol were used. Poojari et al. recovered between
◦
toluene at 70 C over 4 h reaction cycles [32]. In that study fresh
N435 was shown to have lower activity for the first reaction cycle
than in subsequent cycles. It was postulated that swelling of the
acrylic resin allowed for lipase molecules that were immobilized on
the interior of the solid support to become available to the medium
and participate in the ROP of PCL [32].
5
% and 50% residual activity after the polymerization of 1,3-bis(3-
carboxypropyl)-1,1,3,3-tetramethyldisiloxane and 1,4-butanediol,
,6-hexanediol or 1,8-octanediol [13]. The more hydrophobic 1,8-
1
octanediol did not affect the residual activity of the enzyme as
compared to the other diol monomers. Two solvents were used
by these researchers to isolate the acrylic beads in hopes of deter-
mining if there was a solvent effect on the residual activity. When
tetrahydrofuran was the isolation solvent the enzyme retained
a higher amount of activity compared to the enzyme that was
isolated with toluene. The Gross group reported the retention of
approximately 80% of the residual activity from a batch of N435
The increased thermal tolerance of the immobilized lipase can
be potentially attributed to the favourable interaction between
the solid support and the enzyme, or the interaction between
the enzyme and the hydrophobic disiloxanes. The lipase is poten-
tially protected from thermal denaturation due to many favourable,
although relatively weak, interactions it may have with the solid
support (i.e. hydrogen bonding and van der Waals interactions)
which combine to impart stability to the enzyme [33]. Enzymes
are also known to maintain structural rigidity in organic solvents,
as long as the solvent is hydrophobic and non-polar, which lim-
its molecular motion and in turn thermal denaturation [34]. Both
of these factors are under consideration for the observed residual
activity of N435 after use at higher temperatures. The substrate of
immobilization may form electrostatic interactions with the lipase
imparting a protective effect against denaturation. Even though a
hydrophobic solvent was not used here, per se, the hydrophobicity
◦
after 48 h of polymerizing adipic acid and 1,8-octanediol at 70 C
[
14].
There appears to be a significant relationship between the
hydrophobicity of the monomers and the amount of residual enzy-
matic activity of the enzyme catalyst. This is perhaps not surprising
given the native environment in which lipases have evolved to
function. Silicones are known to be some of the most hydrophobic
materials, and as such disiloxane polyesters should offer a suit-
able environment for lipases to function. In fact Candida rugosa
lipase entrapped within a silicone elastomer retained high activity
over extended periods of time even when stored at room temper-
ature [30,31]. Furthermore, the free form of the lipase displayed
higher activity when assayed in silicone oil (D5) compared to simple
hydrocarbons [30,31].
3.5
.0
110
1
9
8
7
6
5
4
00
0
3
0
3.5. Repeated use of N435
2.5
2.0
1.5
1.0
0.5
0.0
0
In light of the residual activity of N435, even after prolonged
0
exposure to elevated temperatures, it was of interest to determine
the long-term thermal tolerance of a single batch of the immo-
bilized catalyst. We were interested to determine the number of
reaction cycles that a single batch of N435 could be used in before
it became unsuitable for catalysis. To accomplish this, a series of
0
0
30
2
1
0
0
0
◦
2
4 h reaction cycles at 100 C were performed. The change in both
the second order rate constant and the degree of monomer con-
version were used to determine the effect of long term reaction
sequences. Between each consecutive use of the catalyst, the reac-
tion mixture was cooled to room temperature and washed with
diethyl ether to cleanse the acrylic beads of any residual polymer.
The acrylic beads were separated from the crude reaction mixture
by filtering through a medium porosity glass fritted filter.
0
1
2
3
4
5
6
7
8
9
10 11
Reaction cycle
Fig. 8. The effect of multiple reaction cycles for a single batch of N435. The apparent
rate constant (open circles) and total monomer conversion after 24 h (black circles)
◦
are shown for each of ten consecutive 24 h reaction cycles at 100 C.

M.B. Frampton et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 149–155
155
of the disiloxanes could be hypothesized to function in a similar
role, and contribute to the stabilization of the lipase.
References
[
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. Conclusions
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◦
◦
Increases in the reaction temperature from 35 C to 100 C led to
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◦
2
4 h at 150 C the enzyme had lost all catalytic activity however it is
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The authors would like to thank Tim Jones (Brock University)
for assistance in acquiring all mass spectrometry data. Funding for
this project was provided by an NSERC Engage grant to PMZ. MBF
was supported by graduate scholarships from the Ontario Gradu-
ate Scholarship, the Ontario Graduate Scholarship in Science and
Technology Programs and the Faculty of Graduate Studies. JPS was
supported by the Brock University Experience Works Program. DM
was supported by Brock University Faculty of Graduate Studies.
TAH is supported by funding provided by NSERC.
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